The invention relates to novel nucleic acid sequences encoding peptide transporters, to novel polypeptides and drug delivery systems.
Peptide transport, a phenomenon defined as the translocation of peptides across the plasma membrane in an energy-dependent manner, has been well documented in bacteria, plants, fungi, and mammals (for reviews see Becker and Naider, 1995; Payne and Smith, 1994). Upon internalization, peptides are quickly hydrolyzed into their amino acid components to serve as sources of amino acids or nitrogen. In addition to acquiring nutrients from the environment, peptide transport has been shown to play a role in recycling cell wall peptides and in transducing signals for group behaviors such as sporulation and competency in B. subtilis and chemotaxis in E. coli. Recently it has been proposed that in Salmonella typhimurium peptide transporters aid the bacteria in evading the host immune response by transporting membrane disrupting peptides away from the plasma membrane (Parra-Lopez et al., 1993). Similarly, in Streptococcus pneumoniae the peptide transporters encoded by plpA and the amiA loci play a role in virulence by modulating adherence to epithelial and endothelial cells (Cundel et al., 1995).
A family of di-/tripeptide transporters named the PTR (Peptide TRansport) Family has recently been identified. This family is characterized by several conserved motifs, has twelve putative transmembrane domains, and is driven by the proton motive force. Members of the PTR family have been identified in a broad variety of eukaryotes and one prokaryote as well (Steiner et al, 1995). Well characterized members of the PTR family are the di- and tripeptide transporters from S. cerevisiae (ScPTR2, Perry et al., 1994) and from C. albicans (CaPTR2, Basrai et al., 1995). Both CaPTR2 and ScPTR2 have been shown to be regulated by nitrogen source and inducible by micromolar amounts of amino acids; their encoded proteins have broad substrate specificities with a preference for peptides containing hydrophobic residues (Basrai et al., 1992; Island et al., 1987). Prior to the establishment of the PTR family, all peptide transporters cloned were from prokaryotes and were members of the ATP Binding Cassette (ABC) Superfamily (Higgins, 1992). Recently, transporters from the PTR family have been identified in the prokaryote Lactococcus lactis (Hagting et al., 1995). However, in eukaryotes all peptide transporters thus far identified are members of the PTR family.
In addition to the di-/tripeptide transporter (CaPTR2) in C. albicans, three observations indicated the existence of another distinct peptide transport system. The first observation was that mutants resistant to the toxic peptide analogs bacilysin, polyoxin, and nikkomycin Z (all demonstrated substrates of the di-/tripeptide transport system) were able to transport tetra- and pentapeptides at wild type levels, and, conversely, mutants resistant to various toxic tetrapeptides were able to transport dipeptides at wildtype levels (Payne and Shallow, 1985; Milewski et al., 1988; McCarthy et al., 1985). Secondly, peptide uptake experiments with radiolabeled compounds and chromophoric substrates demonstrated that dipeptides did not compete with tetra- and pentapeptides for entry into the cell, and vice versa tetra- and pentapeptides did not compete with labeled dipeptides (Milewski et al., 1988; McCarthy et al., 1985; Yadan et al., 1984).. Thirdly, sensitivity to toxic di- and tripeptides was influenced by nitrogen source and micromolar amounts of amino acids while sensitivity to toxic tetra- and pentapeptides was not regulated by similar means (Basrai et al., 1992). The invention described herein relates to the cloning of a novel oligopeptide transporter from C. albicans that does not code for an ABC- or PTR-type transporter.
The invention relates to a new transporter system: a system to transport oligopeptides as opposed to lower peptides. The invention relates to an oligopeptide transporter competent to transport higher oligopeptides, especially tetra- and pentapeptides. The expression of transport activity is evidenced in a heterologous host suggesting that the transporter is an integral membrane transporter. The ability to transport peptides of a size larger than di/tripeptides is highly significant in that it will permit the delivery of greater variety of biological molecules in molecular structure and size into the selected target.
Furthermore, there is provided a novel peptide transport gene from Candida albicans through heterologous expression in Saccharomyces cerevisiae, which encodes an oligopeptide transporter OPT, which is different from the previously identified family of di-/tripeptide transporters named the PTR (Peptide Transport) family. The gene encoding OPT1 appears to constitute the first identified member of a new family of oligopeptide transporters.
The gene sequence revealed the presence of two ORFs separated by a type II intron, and encoding a hydropholic protein of 783 amino acids with an apparent molecular mass of 88 kDa and a pI of 7.1. The size and hydrophobic nature of the predicted protein of OPT1 suggest a membrane/bound protein with at least 12 putative transmembrane domains of 20-24 amino and residues. Findings made in connection with invention indicate that OPT1 is not a member of the PTR or ABC families of membrane transporters.
Peptide utilization mediated by OPT1 showed its ability to mediate the uptake of Lys-Leu-Gly (KLG), Lys-Leu-Leu-Gly (KLLG) SEQ ID NO:6, Lys-Leu-Gly-Leu (KLGL) SEQ ID NO:7 and Lys-Leu-Leu-Leu-Gly (KLLLG) SEQ ID NO:8.
Various eukaryotic transformants of the yeasts are made available by the invention.
The invention provides peptide transporters as a means to facilitate the uptake of otherwise nonpermeating biologically active molecules of medical significance, such as antifingal compounds.
Heretofore it was known that C. albicans can transport and utilize small peptides. The invention allows using C. albicans with the novel oligopeptide transporter for the uptake of peptide-drug adducts. In the search for effective antimicrobial drugs, substances are often found that display toxicity towards intracellular targets when tested in cell-free systems, but are inactive with intact organisms. Frequently this occurs because the potentially toxic agent is impermeable. The invention provides a drug delivery system whereby a toxic moiety is linked or otherwise carried by a molecule which will be taken up and actively transported through a specific permease for delivery to the target. But for the membrane transporter system described herein, such drug delivery system are known. For instance, the uptake by dipeptides containing N3-(4-methoxyfamaroyl)-L-2,3-diamino-propanoic acid (FMDP) has been extensively studied. Literature references dealing with such drug delivery designs are incorporated herein by reference. For instance, it is known that N-acylation can stabilize the carrier toxic agent conjugate to amino peptidase activity. See Peptide Base Drug Design, Becker and Naider cited herein.
Infections attributable to C. albicans are wide spread. The oligopeptide transport system of the invention is useful to deliver anticandidal drugs carried (conjugated or linked or associated) and taken up by the peptide, delivered to the transporter which will deliver it through the membrane to the target.
The oligopeptide transporter of the invention will promote the more effective delivery of anti candidal drugs into organisms infected by C. albicans. Such drugs can be molecules like toxic peptides carried, if necessary by a carrier, or molecules that mimic or are similar in character to the peptides, like peptido-mimetics. The invention also provides for the delivery of the OPT1 gene into a mammalian target call where it will express the oligopeptide transporter, thereby facilitates the targeting of the desired drug.
The ability to use the transport system encoded by the OPT gene will allow delivery of toxic agents specifically into cells or organisms expressing this gene. Thus, if pathogenic fungi express such a gene in an infected human host that is not capable of expressing this gene, then antifungal agents can be designed to kill the invading pathogen without having any adverse effects on the human host. Such non-toxic antifungal agents are the major goal of all pharmaceutical companies with antifungal drug programs. Currently, extensive research is carried out throughout the world in the search for antifungal drugs.
The OPT gene of C. albicans opens the way for gene discovery of a family of plant oligopeptide transport genes. Genes in the family represented by the OPT gene of C. albicans have been found to date only in other fungi, and some potential homologous genes have been noted in the plant EST database. Using fungi as the heterologous hosts for testing oligopeptide transport ability, full-length plant genes should be uncovered and characterized by techniques used in this invention to clone di-/tripeptide plant transporters. Thus, oligopeptides might be useful agents as herbicides or growth stimulators depending on the chemical constituents of a modified oligopeptide. Delivery of such oligopeptide-based analogs to plant cells via the oligopeptide transport system would allow specificity in targeting. Also, uptake into the plant cell would occur in large quantities due to the ability to transport systems to concentrate substrates intracellularly to high levels.
Other embodiments will become apparent from the description that follows.